Development of novel detergents for use in pcr systems

ABSTRACT

This disclosure relates to novel detergents for use in various procedures including, for example, nucleic acid amplification reactions such as polymerase chain reaction (PCR). Methods for preparing the modified detergents are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/225,261, filed Dec. 19, 2018; which is a divisionalapplication of U.S. patent application Ser. No. 15/273,824, filed Sep.23, 2016, now U.S. Pat. No. 10,202,639; which is a divisionalapplication of U.S. patent application Ser. No. 14/250,154, filed Apr.10, 2014, now U.S. Pat. No. 9,493,414; which is a divisional applicationof U.S. patent application Ser. No. 13/492,576, filed Jun. 8, 2012, nowU.S. Pat. No. 8,980,333; which claims the benefit of priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application No. 61/494,812,filed Jun. 8, 2011, entitled “Design and Development of Novel Detergentsfor Use in PCR Systems,” the disclosures of which are incorporatedherein by reference in their entirety.

FIELD

This disclosure relates to modified detergents for use in variousprocedures including, for example, nucleic acid amplification reactionssuch as polymerase chain reaction (PCR). Methods for preparing themodified detergents are also described.

BACKGROUND

Many widely known recombinant DNA techniques involve replicating orpolymerizing and/or amplifying DNA. One such example is the polymerasechain reaction (PCR). During PCR, the reaction cycles repeatedly betweentwo temperatures, a low and a high temperature (e.g., 55° C. and 95° C.)in the presence of a thermostable DNA polymerase enzyme. The totalperiod of time spent at the high temperature over the course of thereaction depends upon the total number of cycles, the duration of thehigh temperature step of each cycle, and the ramp speed (i.e., the rateat which the thermocycler changes from one temperature to another).Although the DNA polymerases used in PCR are highly thermostable, theytend to become inactive at high temperatures over time. Furthermore,these polymerases may also become inactive by being introduced intoreaction mixture environments with sub-optimal concentration ofcofactors, or that have sub-optimal pH levels, or that include thepresence of chemical or biological inhibitors.

One way of stabilizing an enzyme under such conditions is to add astabilizing agent, such as a surfactant. Surfactants, such asdetergents, are surface-active compounds that stabilize the interfacebetween the active form of an enzyme and its liquid environment. Forexample, the activity of Taq DNA polymerase has been stabilized by theaddition of nonionic detergents, such as NP-40 or Tween® 20 (Bachmann,et al. Nuc. Acids Res. 18(5): 1309 (1990)). In some applications,however, Tween® 20-stabilized DNA polymerases have low efficiencies ofamplification or lead to the amplification of non-specific products. Inaddition, some detergents are required a high concentrations. Moreover,some detergents (e.g., NP-40) are also known to have toxic properties.There is a need, therefore, for detergents that improve the stability ofthermostable DNA polymerases in solution, and particularly detergentsthat improve enzyme stability without imparting any of the disadvantagesof currently used detergents.

BRIEF DESCRIPTION OF THE DRAWINGS

All amplification plots shown herein graphically represent targetnucleic acid amplification as ΔRn (y-axis) as a function of cycle number(x-axis).

FIGS. 1A and 1B. Titration of novel detergents Dt1 and Dt2 at differentconcentrations using 1 Kb and 3 Kb PCR products amplified according tocertain exemplary embodiments of the methods and compositions disclosedherein.

FIG. 2. Amplification of the rhodopsin gene in the presence of noveldetergents Dt1 and Dt2 according to certain exemplary embodiments of themethods and compositions disclosed herein.

FIGS. 3A and 3B. Comparison of novel detergent Dt2 at 0.004% and 0.0002%compared to NP-40/Tween® 20 for 0.1-1 Kb PCR products amplifiedaccording to certain exemplary embodiments of the methods andcompositions disclosed herein.

FIGS. 4A and 4B. Comparison of novel detergents Dt2 at 0.004% and 0.002%to NP-40/Tween® 20 for 1-2 Kb PCR products amplified according tocertain exemplary embodiments of the methods and compositions disclosedherein.

FIGS. 5A and 5B. PCR Activity: Comparison of novel detergent Dt2 toBrij-58 alone for rhodopsin gene products amplified according to certainexemplary embodiments of the methods and compositions disclosed herein.

FIG. 6. Titration of novel detergents Dt2 and Dt4 using the Rhod-1043target sequence amplified according to certain exemplary embodiments ofthe methods and compositions disclosed herein.

FIGS. 7A through 7D. Comparison of Dt4 to Tween 20: amplification ofbeta-2 microglobulin (B2M), glyceraldehyde 3-phosphate dehydrogenase(GAPDH), large ribosomal protein (RPLPO), and glucuronidase beta (GUSB)according to certain exemplary embodiments of the methods andcompositions disclosed herein.

FIGS. 8A through 8D. Comparison of Dt4 to Tween 20 (log scale):amplification of B2M, GAPDH, RPLPO, and GUSB according to certainexemplary embodiments of the methods and compositions disclosed herein.

FIG. 9. Comparison of Dt4 to Tween 20: amplification efficiency ofvarious PCR products as represented by Cq according to certain exemplaryembodiments of the methods and compositions disclosed herein.

FIG. 10. Comparison of Dt1, Dt3, Dt5, Dt6, and Dt7 to Tween 20;amplification performed according to certain exemplary embodiments ofthe methods and compositions disclosed herein.

FIGS. 11A and 11B. Amplification plot of amplification reactions ofhypoxanthine phosphoriboxyltransferase (HPRT1) comparing the activity ofDt4 to Brij-58 and Tween® 20 (0.001% and 0.0008% of each) according tocertain exemplary embodiments of the methods and compositions disclosedherein.

FIGS. 12A and 12B. Amplification plot of amplification reactions ofHPRT1 comparing the activity of Dt4 to Brij-58 and Tween® 20 (0.0006%and 0.0004% of each) according to certain exemplary embodiments of themethods and compositions disclosed herein.

FIGS. 13A and 13B. Amplification plot of amplification reactions ofpeptidyl prolyl isomerase A (PPIA) comparing the activity of Dt4 toBrij-58 and Tween® 20 (0.001% and 0.0008% of each) according to certainexemplary embodiments of the methods and compositions disclosed herein.

FIGS. 14A and 14B. Amplification plot of amplification reactions of PPIAcomparing the activity of Dt4 to Brij-58 and Tween® 20 (0.0006% and0.0004% of each) according to certain exemplary embodiments of themethods and compositions disclosed herein.

FIGS. 15A and 15B. Amplification plot of amplification reactions of PPIAcomparing the activity of Dt4 to Brij-58 and Tween® 20 (0.0002% and0.0001% of each) according to certain exemplary embodiments of themethods and compositions disclosed herein.

FIG. 16. Amplification plot of amplification reactions of B2M comparingthe activity of 0.002% Dt4 to 0.01% and Tween® 20 according to certainexemplary embodiments of the methods and compositions disclosed herein.

FIG. 17. Amplification plot of amplification reactions of GAPDHcomparing the activity of 0.002% Dt4 to 0.01% and Tween® 20 according tocertain exemplary embodiments of the methods and compositions disclosedherein.

FIG. 18. Amplification plot of amplification reactions of RPLPOcomparing the activity of 0.002% Dt4 to 0.01% and Tween® 20 according tocertain exemplary embodiments of the methods and compositions disclosedherein.

FIG. 19. Graphical representation of amplification reactions (Cq) takenat approximately one week intervals over two months demonstrating thestability of the polymerase in a 5× buffer (amplification reactions ofACTB (actin-beta), GAPDH, PPIA, and RPLPO) according to certainexemplary embodiments of the methods and compositions disclosed herein.

FIG. 20. Graphical representation of amplification reactions (delta Rn)taken at approximately one week intervals over two months demonstratingthe stability of the polymerase in a 5× buffer (amplification reactionsof ACTB (actin-beta), GAPDH, PPIA, and RPLPO) according to certainexemplary embodiments of the methods and compositions disclosed herein.

FIG. 21. Comparison of two different Dt4 lots with Tween 20 (Cq,amplification reactions of RPLPO, ACTB, PPIA, GAPDH, PGK1(phosphoglycerate kinase 1), B2M, GUSB, and HPRT1) Graphicalrepresentation of amplification reactions (Cq) taken at approximatelyone week intervals over two months demonstrating the stability of thepolymerase in a 5× buffer (amplification reactions of ACTB (actin-beta),GAPDH, PPIA, and RPLPO) according to certain exemplary embodiments ofthe methods and compositions disclosed herein.

FIG. 22. Comparison of two different Dt4 lots with Tween 20 (Delta Rn,amplification reactions of RPLPO, ACTB, PPIA, GAPDH, PGK1(phosphoglycerate kinase 1), B2M, GUSB, and HPRT1) Graphicalrepresentation of amplification reactions (Cq) taken at approximatelyone week intervals over two months demonstrating the stability of thepolymerase in a 5× buffer (amplification reactions of ACTB (actin-beta),GAPDH, PPIA, and RPLPO) according to certain exemplary embodiments ofthe methods and compositions disclosed herein.

FIG. 23. Comparison of Dt4 amplification across a variety of TaqManassays, according to certain exemplary embodiments of the methods andcompositions disclosed herein.

SUMMARY

Provided herein are modified detergents for a variety of uses, includingbut not limited to nucleic acid amplification reactions. In someembodiments, ionic and zwitterionic detergents are synthesized bychemically modifying the simple starting materials are provided. All theintermediates were observed and analyzed using LC-MS analysis and werethen subsequently used without purifications. In some embodiments, noveldetergents such as Dt4 (described below), are provided. These noveldetergents may be used in a variety of procedures including, forexample, nucleic acid amplification reactions such as polymerase chainreaction (PCR).

In certain embodiments, the modified detergents have the followingstructural formula:

wherein:

-   -   R¹ is H, (C₁-C₃₀) alkyl, (C₁-C₃₀) substituted alkyl, (C₁-C₃₀)        heteroalkyl, (C₁-C₃₀) substituted heteroalkyl, aryl, substituted        aryl, heteroaryl, substituted heteroaryl, phenyl, substituted        phenyl, where the substituted aryl or substituted phenyl is        substituted by at least one (C₁-C₃₀) alkyl, (C₁-C₃₀) substituted        alkyl, (C₁-C₃₀) heteroalkyl, or (Ci-C3o) substituted        heteroalkyl;    -   R² and R³ are each independently H, CH₃, CH(CH₃)₂,CH₂(C₆H₅), or        C(CH₃)₃; R⁴ and R⁵ are each independently H, CH₃, CH(CH₃)₂,C₆H₅,        CH₂(C₆H₅), C(CH₃)₃, CH₂CH(CH₃)₂,CHCH₂CH(CH₃)₂,CH₂C₆H₅OH, CH₂C═CH        NH(C₆H₅), CH₂C═CHN═CHNH, CH₂COOH,        CH₂CONH₂,(CH₂)₂CONH₂,(CH₂)₂COOH,CH₂SH, (CH₂)_(n)NH, (CH₂)_(n)N,        CH₂OH, CH(OH)CH₃, (CH₂)₂SCH₃, (CH₂)₃NHC(NH₂)═NH, or,        alternatively, R³ is taken together with R⁵ to form a 5- or        6-membered ring which is optionally substituted with at least        one (C₁-C₃₀) alkyl, (C₁-C₃₀) substituted alkyl, (C₁-C₃₀)        heteroalkyl, (C₁-C₃₀) substituted heteroalkyl; and,    -   each n is independently any positive integer, including but not        limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, R¹ is a C₈ alkyl. In certain embodiments, R¹ isa C₁₆ alkyl. In certain embodiments, R² and R³ are each independentlyselected from H and CH₃. In certain embodiments, R⁴ and R⁵ are eachindependently selected from H, (CH₂)_(n)NH, (CH₂)_(n)N, oralternatively, R³ is taken together with R⁵ to form a 5- or 6-memberedring.

Also provided are the methods for polymerizing and/or amplifying anucleic acid comprising mixing a target nucleic acid with at least onepolymerase, a primer, dNTPs, and at least one novel detergent of FormulaI, and polymerizing and/or amplifying the target nucleic acid. In someembodiments of such methods, at least one primer is utilized. In certainembodiments, a nucleic acid amplification reaction mixture comprising atleast one polymerase, dNTPs, at least one primer, and at least one noveldetergent of Formula I is provided. In some embodiments, the reactionmixture may further comprise a detectable label. In certain embodiments,the methods further include one or more steps for detecting thedetectable label to quantitate the amplified nucleic acid. In certainembodiments, methods for inhibiting inactivation of a polymerase duringa thermal cycling process by including therein a novel detergent ofFormula I are provided. In certain embodiments, methods for providing anenzyme having polymerase activity and at least one novel detergent ofFormula I and combining the same to form a mixture under conditions suchthat the polymerase activity of the enzyme is stabilized are provided.In certain embodiments, the polymerase is thermostable. In certainembodiments, the methods described herein provide amplificationreactions with amplification efficiency similar to (e.g., approximatelythe same), or increased amplification efficiency when in the presence ofa conventional (e.g., known) detergent such as, for example, NP-40and/or Tween® 20. In some embodiments, the novel detergents describedherein may substitute for NP-40 and/or Tween® 20 in an amplificationreaction.

In certain embodiments, the effective concentration of the at least onenovel detergent described herein in a reaction mixture is less than thatrequired of conventional detergents such as NP-40 and/or Tween® 20. Insome such embodiments, the effective concentration of the at least onenovel detergent(s) in a reaction mixture may be up to, about, or atleast one, two, three, four, five, six, seven, eight, nine, or ten timesless than that required of conventional detergent(s) such as NP-40and/or Tween® 20. Methods for producing the novel detergents are alsoprovided.

In certain embodiments, compositions comprising at least one of thenovel detergents of Formula I are provided herein. In certainembodiments, compositions comprising at least one polymerase and atleast one of the novel detergents of Formula I are provided. In someembodiments, the polymerase is thermostable. Kits comprising reagentsand the like necessary to carry out such methods or prepare suchmixtures are also provided.

DETAILED DESCRIPTION

Provided herein are novel detergents for a variety of uses, includingbut not limited to nucleic acid polymerization and/or amplificationreactions. In some embodiments, ionic and zwitterionic detergents aresynthesized chemically utilizing simpler starting materials areprovided. In some embodiments, the novel detergents such as Dt1,Dt₂,Dt3, Dt4, Dt5, Dt6, Dt7, Dt8, Dt9, and Dt10 (described below) areprovided. These novel detergents may be used in a variety of proceduresincluding, for example, nucleic acid polymerization and/or amplificationreactions such as the polymerase chain reaction (PCR). In someembodiments, the presence of one or more of the novel detergents ofFormula I may stabilize the polymerase within a reaction mixture,decrease inhibition of the polymerase within a reaction mixture, and/orincrease the polymerization and/or amplification efficiency of thepolymerase. As such, reaction mixtures comprising at least onepolymerase and at least one of the novel detergents of Formula I areprovided. Such reaction mixtures may further comprise one or more dNTPsand at least one nucleic acid amplification primer (e.g., PCR primer).

In certain embodiments, compositions comprising at least one of thenovel detergents of Formula I are provided herein. In certainembodiments, compositions comprising at least one polymerase and atleast one of the novel detergents of Formula I are provided. In someembodiments, the polymerase is thermostable. Kits comprising thecomponents of such reaction mixtures and optionally also other reagentsnecessary for carrying out such methods or for preparing such mixturesare also provided.

Novel detergents and methods of preparing and using the same aredescribed herein. The term “novel detergent” typically refers to adetergent of Formula I. In certain embodiments, the term “detergent” mayrefer to one or more novel detergents, optionally including one or more“conventional detergents.” As used herein, the term “conventionaldetergent” refers to a detergent other than those described herein underFormula I. In some embodiments, the term “detergent” may refer to anovel detergent only, or a combination of one or more novel detergentswith one or more conventional detergents. Similarly, the use of the term“at least one novel detergent” may refer to one or more novel detergentsalone, with another novel detergent, and/or with one or moreconventional detergents. Thus, in some embodiments, the compositionsand/or reaction mixtures described herein may further comprise one ormore conventional detergents such as, for example and withoutlimitation, a nonionic detergent, Brij-58, CHAPS,n-Dodecyl-b-D-maltoside, NP-40, sodium dodecyl sulphate (SDS), TRITON®X-15, TRITON® X-35, TRITON® X-45, TRITON® X-100, TRITON® X-102, TRITON®X-114, TRITON® X-165, TRITON® X-305, TRITON® X-405, TRITON® X-705,Tween® 20 and/or ZWITTERGENT®. Other detergents may also be suitable, asmay be determined by one of skill in the art (see, e.g., U.S. PatentApplication Publication No. 2008/0145910; U.S. Patent ApplicationPublication No. 2008/0064071; U.S. Pat. No. 6,242,235; U.S. Pat. No.5,871,975; and U.S. Pat. No. 6,127,155 for exemplary detergents; all ofwhich are hereby incorporated herein by reference in their entirety.)Additional detergents may also be suitable, as would be determined bythe skilled artisan.

In certain embodiments, the novel detergents have the followingstructural formula:

wherein:

-   -   R¹ is H, (C₁-C₃₀) alkyl, (C₁-C₃₀) substituted alkyl, (C₁-C₃₀)        heteroalkyl, (C₁-C₃₀) substituted heteroalkyl, aryl, substituted        aryl, heteroaryl, substituted heteroaryl, phenyl, substituted        phenyl, where the substituted aryl or substituted phenyl is        substituted by at least one (C₁-C₃₀) alkyl, (C₁-C₃₀) substituted        alkyl, (C₁-C₃₀) heteroalkyl, or (C₁-C₃₀) substituted        heteroalkyl;    -   R² and R³ are each independently H, CH₃, CH(CH₃)₂,CH₂(C₆H₅), or        C(CH₃)₃;    -   R⁴ and R⁵ are each independently H, CH₃, CH(CH₃)₂,C₆H₅,        CH₂(C₆H₅), C(CH₃)₃, CH₂CH(CH₃)₂,CHCH₂CH(CH₃)₂,CH₂C₆H₅OH, CH₂C═CH        NH(C₆H₅), CH₂C═CHN═CHNH, CH₂COOH,        CH₂CONH₂,(CH₂)₂CONH₂,(CH₂)₂COOH,CH₂SH, (CH₂)_(n)NH, (CH₂)_(n)N,        CH₂OH, CH(OH)CH₃, (CH₂)₂SCH₃, (CH₂)₃NHC(NH₂)═NH, or,        alternatively, R³ is taken together with R⁵ to form a 5- or        6-membered ring which is optionally substituted with at least        one (C₁-C₃₀) alkyl, (C₁-C₃₀) substituted alkyl, (C₁-C₃₀)        heteroalkyl, (C₁-C₃₀) substituted heteroalkyl; and,    -   each n is independently any positive integer, including but not        limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, R¹ is a C₈ alkyl. In certain embodiments, R¹ isa C₁₆ alkyl. In certain embodiments, R² and R³ are each independentlyselected from H and CH₃. In certain embodiments, R⁴ and R⁵ are eachindependently selected from H, (CH₂)_(n)NH, (CH₂)_(n)N, oralternatively, R³ is taken together with R⁵ to form a 5- or 6-memberedring.

The detergents of Formula I may be prepared by utilizing simplerstarting compound material to provide new and/or improved detergents andits properties thereto. The intermediates were analyzed using LC-MSanalysis and used without purification to perform the next step.

A method for preparing the novel detergents described herein includessequentially combining Compound A (as shown in Process 1) (e.g., 1 eq),methyltriphenoxyphosphonium iodide (e.g., 4 eq.), and N,N-dimtheylformamide (DMF) (e.g., 6 mL) to an aluminum foil covered round bottomflask (e.g., 50 mL). The reaction is then stirred for a sufficientperiod of time (e.g., 3 days) under an appropriate atmosphere (e.g.,argon) at an appropriate temperature (e.g., room temperature). After thesufficient period of time (e.g., 3 days), progress of the reaction maybe monitored (e.g., using analytical liquid chromatography/massspectrometry (LC-MS). The appearance of an intermediate product patternmay confirm formation of the modified detergent. This expectedintermediate product (Intermediate B) may or may not (typical) beisolated. To this intermediate product, amino acid ester hydrochloridesalt (e.g., 2 eq.) and Et₃N (e.g., 2 eq.) may be added. The reactionmixture may be heated for an appropriate period of time (e.g., 3-4 days)at an appropriate temperature (e.g., 65° C.). The progress of thereaction may be monitored using analytical LC-MS. The reaction mixtureis then typically cooled to an appropriate temperature (e.g., roomtemperature) and concentrated (e.g., on a rotovapor) to appropriatevolume (e.g., approximately 2 mL). The concentrated crude mixture maythen be purified by preparative HPLC. The desired fractions may then bepooled and concentrated (e.g., on the rotovapor) to afford the desiredproduct (e.g., as in Process 1 to produce the ionic detergents Dt1, Dt3,Dt5, Dt7, Dt9, Dt11, and Dt12). This product may then be subjected to ahydrolysis reaction (e.g., using 2N NaOH). The reaction mixture may thenbe stirred (e.g., at room temperature) until all the starting materialis consumed as determined by analytical LC-MS. This may be followed byneutralization (e.g., with Amberlite) to produce the zwitterionic finalproducts (e.g., Dt₂,Dt4, Dt6, Dt10) or anionic final product (Dt8).

Thus, an exemplary method for the development of the novel detergents ofFormula I is shown below:

where R², R³, R⁴, R⁵ and n are as described hereinabove, and X isselected from the group consisting of H, CH₃, CH₂CH₃, CH₂(C₆H₅), andC(CH₃)₃. The novel detergents of Formula I may be, for example, ionic(e.g., cationic, anioinic, zwitterionic). Exemplary novel detergentsmade using Process 1 are shown below:

where n is as described hereinabove. In certain embodiments, each n isindependently 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30.

In certain embodiments methods for polymerizing and/or amplifying anucleic acid comprising mixing a target nucleic acid with at least onepolymerase, a primer, dNTPs, and at least one novel detergent of FormulaI, and polymerizing and/or amplifying the target nucleic acid areprovided. In certain embodiments, the methods may include at least oneprimer. In certain embodiments, a nucleic acid amplification reactionmixture comprising at least one polymerase, dNTPs, at least one primer,and at least one novel detergent of Formula I is provided. In certainembodiments, the reaction mixture may further comprise a detectablelabel. In certain embodiments, the methods include one or more steps fordetecting and/or quantitating the detectable label to detect and/orquantitate the amplified nucleic acid.

In certain embodiments methods for inhibiting inactivation of apolymerase during a thermal cycling process by including therein a noveldetergent of Formula I are provided. In certain embodiments, methods forproviding an enzyme having polymerase activity and at least one noveldetergent of Formula I and combining the same to form a mixture underconditions such that the polymerase activity of the enzyme is stabilizedare provided. In certain embodiments, the polymerase is thermostable. Incertain embodiments, the polymerase is thermostable. In certainembodiments, the methods described herein provide amplificationreactions with amplification efficiency similar to (e.g., approximatelythe same), or increased amplification efficiency when in the presence ofa conventional (e.g., known) detergent such as, for example, NP-40and/or Tween® 20. In some embodiments, the novel detergents describedherein may substitute for NP-40 and/or Tween® 20 in an amplificationreaction.

In certain embodiments, the “effective concentration” (e.g., the amountthat will support an amplification reaction such as PCR) of the at leastone novel detergent of Formula I (e.g., Dt1, Dt₂, Dt3, Dt4, Dt5, Dt6,Dt7, Dt8, Dt9, Dt10, Dt11 and/or Dt 12) in a reaction mixture may behigher, the same, or lower than that required of conventional detergents(e.g., NP-40 and/or Tween® 20). In some such embodiments, the effectiveconcentration of the at least one novel detergent(s) (e.g., Dt4) in areaction mixture may be up to about or at least one, two, three, four,five, six, seven, eight, nine, or ten times less than that required ofconventional detergent(s) such as NP-40 and/or Tween® 20. For example,NP-40 or Tween® 20 are typically included in a reaction at about 0.01%or less (e.g., as determined by dilution from a stock solution into areaction mixture). The novel detergents described herein may, in certainembodiments, be used at a lower concentration (e.g., as a percentage(i.e., w/v or v/v)) than conventional detergents (e.g., 0.002% for Dt4as compared to 0.01% Tween 20; FIGS. 11A and 11B, 12A and 12B, 13A and13B, 14A and 14B, 15A and 15B, and 16 through 18).

In certain embodiments, methods for polymerizing and/or amplifying anucleic acid comprising mixing a nucleic acid of interest (e.g., atarget nucleic acid) with at least one polymerase, a primer, dNTPs, andat least one novel detergent of Formula I, and polymerizing and/oramplifying the target nucleic acid are provided. In certain embodiments,the methods include at least one primer. In certain embodiments, anucleic acid amplification reaction mixture(s) comprising at least onepolymerase, dNTPs, at least one primer, and at least one modifieddetergent of Formula I is provided. In other embodiments, methods forusing such mixture(s) are provided. Target nucleic acids may beamplified using any of a variety of reactions and systems.

As used herein, the terms “amplification”, “nucleic acid amplification”,or “amplifying” refer to the production of multiple copies of a nucleicacid template, or the production of multiple nucleic acid sequencecopies that are complementary to the nucleic acid template. The terms(including the term “polymerizing”) may also refer to extending anucleic acid template (e.g., by polymerization). The amplificationreaction may be a polymerase-mediated extension reaction such as, forexample, a polymerase chain reaction (PCR). However, any of the knownamplification reactions may be suitable for use as described herein. Theterm “amplifying” that typically refers to an “exponential” increase intarget nucleic acid may be used herein to describe both linear andexponential increases in the numbers of a select target sequence ofnucleic acid.

The term “amplification reaction mixture” and/or “master mix” may referto an aqueous solution comprising the various (some or all) reagentsused to amplify a target nucleic acid. Such reactions may also beperformed using solid supports (e.g., an array). The reactions may alsobe performed in single or multiplex format as desired by the user. Thesereactions typically include enzymes, aqueous buffers, salts,amplification primers, target nucleic acid, and nucleosidetriphosphates. Depending upon the context, the mixture can be either acomplete or incomplete amplification reaction mixture. The method usedto amplify the target nucleic acid may be any available to one of skillin the art. Any in vitro means for multiplying the copies of a targetsequence of nucleic acid may be utilized. These include linear,logarithmic, and/or any other amplification method. While thisdisclosure may generally discuss PCR as the nucleic acid amplificationreaction, it is expected that the modified detergents describe hereinshould be effective in other types of nucleic acid amplificationreactions, including both polymerase-mediated amplification reactions(such as helicase-dependent amplification (HDA), recombinase-polymeraseamplification (RPA), and rolling chain amplification (RCA)), as well asligase-mediated amplification reactions (such as ligase detectionreaction (LDR), ligase chain reaction (LCR), and gap-versions of each),and combinations of nucleic acid amplification reactions such as LDR andPCR (see, for example, U.S. Pat. No. 6,797,470). For example, themodified detergents may be used in, for example, variousligation-mediated reactions, where for example ligation probes areemployed as opposed to PCR primers. Additional exemplary methods includepolymerase chain reaction (PCR; see, e.g., U.S. Pat. Nos. 4,683,202;4,683,195; 4,965,188; and/or 5,035,996), isothermal procedures (usingone or more RNA polymerases (see, e.g., PCT Publication No. WO2006/081222), strand displacement (see, e.g., U.S. Pat. No. RE39007E),partial destruction of primer molecules (see, e.g., PCT Publication No.WO 2006/087574)), ligase chain reaction (LCR) (see, e.g., Wu, et al.,Genomics 4: 560-569 (1990)), and/or Barany, et al. Proc. Natl. Acad.Sci. USA 88:189-193 (1991)), Qβ RNA replicase systems (see, e.g., PCTPublication No. WO 1994/016108), RNA transcription-based systems (e.g.,TAS, 3SR), rolling circle amplification (RCA) (see, e.g., U.S. Pat. No.5,854,033; U.S. Patent Application Publication No. 2004/265897; Lizardiet al. Nat. Genet. 19: 225-232 (1998); and/or Bailer et al. Nucleic AcidRes., 26: 5073-5078 (1998)), and strand displacement amplification (SDA)(Little, et al. Clin. Chem. 45:777-784 (1999)), among others. Thesesystems, along with the many other systems available to the skilledartisan, may be suitable for use in polymerizing and/or amplifyingtarget nucleic acids for use as described herein.

“Amplification efficiency” may refer to any product that may bequantified to determine copy number (e.g., the term may refer to a PCRamplicon, an LCR ligation product, and/or similar product). Whether aparticular detergent functions as desired in a particular amplificationreaction may be determined by carrying out at least two separateamplification reactions, each reaction being carried out in the absenceand presence, respectively, of a detergent quantifying amplificationthat occurs in each reaction. Various concentrations or combinations ofdetergents may also be tested in separate reaction mixtures to determinethe effect on amplification efficiency. The amplification and/orpolymerization efficiency may be determined by various methods known inthe art, including, but not limited to, determination of calibrationdilution curves and slope calculation, determination using qBasesoftware as described in Hellemans et al., Genome Biology 8:R19 (2007),determination using the delta delta Cq (AACq) calculation as describedby Livak and Schmittgen, Methods 25:402 (2001), or by the method asdescribed by Pfaffl, Nucl. Acids Res. 29:e45 (2001), all of which areherein incorporated by reference in their entirety.

Exemplary methods for polymerizing and/or amplifying nucleic acidsinclude, for example, polymerase-mediated extension reactions. Forinstance, the polymerase-mediated extension reaction can be thepolymerase chain reaction (PCR). In other embodiments, the nucleic acidamplification reaction is a multiplex reaction. For instance, exemplarymethods for polymerizing and/or amplifying and detecting nucleic acidssuitable for use as described herein are commercially available asTaqMan® (see, e.g., U.S. Pat. Nos. 4,889,818; 5,079,352; 5,210,015;5,436,134; 5,487,972; 5,658,751; 5,210,015; 5,487,972; 5,538,848;5,618,711; 5,677,152; 5,723,591; 5,773,258; 5,789,224; 5,801,155;5,804,375; 5,876,930; 5,994,056; 6,030,787; 6,084,102; 6,127,155;6,171,785; 6,214,979; 6,258,569; 6,814,934; 6,821,727; 7,141,377; and/or7,445,900, all of which are hereby incorporated herein by reference intheir entirety). TaqMan® assays are typically carried out by performingnucleic acid amplification on a target polynucleotide using a nucleicacid polymerase having 5′-to-3′ nuclease activity, a primer capable ofhybridizing to said target polynucleotide, and an oligonucleotide probecapable of hybridizing to said target polynucleotide 3′ relative to saidprimer. The oligonucleotide probe typically includes a detectable label(e.g., a fluorescent reporter molecule) and a quencher molecule capableof quenching the fluorescence of said reporter molecule. Typically, thedetectable label and quencher molecule are part of a single probe. Asamplification proceeds, the polymerase digests the probe to separate thedetectable label from the quencher molecule. The detectable label (e.g.,fluorescence) is monitored during the reaction, where detection of thelabel corresponds to the occurrence of nucleic acid amplification (e.g.,the higher the signal the greater the amount of amplification).Variations of TaqMan® assays (e.g., LNA™ spiked TaqMan® assay) are knownin the art and would be suitable for use in the methods describedherein.

Another exemplary system suitable for use as described herein utilizesdouble-stranded probes in displacement hybridization methods (see, e.g.,Morrison et al. Anal. Biochem., 18:231-244 (1989); and/or Li, et al.Nucleic Acids Res., 30(2,e5) (2002)). In such methods, the probetypically includes two complementary oligonucleotides of differentlengths where one includes a detectable label and the other includes aquencher molecule. When not bound to a target nucleic acid, the quenchersuppresses the signal from the detectable label. The probe becomesdetectable upon displacement hybridization with a target nucleic acid.Multiple probes may be used, each containing different detectablelabels, such that multiple target nucleic acids may be queried in asingle reaction.

Additional exemplary methods for polymerizing and/or amplifying anddetecting target nucleic acids suitable for use as described hereininvolve “molecular beacons”, which are single-stranded hairpin shapedoligonucleotide probes. In the presence of the target sequence, theprobe unfolds, binds and emits a signal (e.g., fluoresces). A molecularbeacon typically includes at least four components: 1) the “loop”, an18-30 nucleotide region which is complementary to the target sequence;2) two 5-7 nucleotide “stems” found on either end of the loop and beingcomplementary to one another; 3) at the 5′ end, a detectable label; and4) at the 3′ end, a quencher moiety that prevents the detectable labelfrom emitting a single when the probe is in the closed loop shape (e.g.,not bound to a target nucleic acid). Thus, in the presence of acomplementary target, the “stem” portion of the beacon separates outresulting in the probe hybridizing to the target. Other types ofmolecular beacons are also known and may be suitable for use in themethods described herein. Molecular beacons may be used in a variety ofassay systems. One such system is nucleic acid sequence-basedamplification (NASBA)®, a single step isothermal process forpolymerizing and/or amplifying RNA to double stranded DNA withouttemperature cycling. A NASBA reaction typically requires avianmyeloblastosis virus (AMV), reverse transcriptase (RT), T7 RNApolymerase, RNase H, and two oligonucleotide primers. Afteramplification, the amplified target nucleic acid may be detected using amolecular beacon. Other uses for molecular beacons are known in the artand would be suitable for use in the methods described herein.

The Scorpions™ system is another exemplary assay format that may be usedin the methods described herein. Scorpions™ primers are bi-functionalmolecules in which a primer is covalently linked to the probe, alongwith a detectable label (e.g., a fluorophore) and a non-detectablequencher moiety that quenches the fluorescence of the detectable label.In the presence of a target nucleic acid, the detectable label and thequencher separate which leads to an increase in signal emitted from thedetectable label. Typically, a primer used in the amplification reactionincludes a probe element at the 5′ end along with a “PCR blocker”element (e.g., a hexaethylene glycol (HEG) monomer (Whitcombe, et al.Nat. Biotech. 17: 804-807 (1999)) at the start of the hairpin loop. Theprobe typically includes a self-complementary stem sequence with adetectable label at one end and a quencher at the other. In the initialamplification cycles (e.g., PCR), the primer hybridizes to the targetand extension occurs due to the action of polymerase. The Scorpions™system may be used to examine and identify point mutations usingmultiple probes that may be differently tagged to distinguish betweenthe probes. Using PCR as an example, after one extension cycle iscomplete, the newly synthesized target region will be attached to thesame strand as the probe. Following the second cycle of denaturation andannealing, the probe and the target hybridize. The hairpin sequence thenhybridizes to a part of the newly produced PCR product. This results inthe separation of the detectable label from the quencher and causesemission of the signal. Other uses for such labeled probes are known inthe art and would be suitable for use in the methods described herein.

The nucleic acid polymerases that may be employed in the disclosednucleic acid amplification reactions may be any that function to carryout the desired reaction including, for example, a prokaryotic, fungal,viral, bacteriophage, plant, and/or eukaryotic nucleic acid polymerase.As used herein, the term “DNA polymerase” refers to an enzyme thatsynthesizes a DNA strand de novo using a nucleic acid strand as atemplate. DNA polymerase uses an existing DNA or RNA as the template forDNA synthesis and catalyzes the polymerization of deoxyribonucleotidesalongside the template strand, which it reads. The newly synthesized DNAstrand is complementary to the template strand. DNA polymerase can addfree nucleotides only to the 3′-hydroxyl end of the newly formingstrand. It synthesizes oligonucleotides via transfer of a nucleosidemonophosphate from a deoxyribonucleoside triphosphate (dNTP) to the3′-hydroxyl group of a growing oligonucleotide chain. This results inelongation of the new strand in a 5′-to-3′ direction. Since DNApolymerase can only add a nucleotide onto a pre-existing 3′-OH group, tobegin a DNA synthesis reaction, the DNA polymerase needs a primer towhich it can add the first nucleotide. Suitable primers may compriseoligonucleotides of RNA or DNA, or chimeras thereof (e.g., RNA/DNAchimerical primers). The DNA polymerases may be a naturally occurringDNA polymerases or a variant of natural enzyme having theabove-mentioned activity. For example, it may include a DNA polymerasehaving a strand displacement activity, a DNA polymerase lacking 5′-to-3′exonuclease activity, a DNA polymerase having a reverse transcriptaseactivity, or a DNA polymerase having an endonuclease activity.

Suitable nucleic acid polymerases may also comprise holoenzymes,functional portions of the holoenzymes, chimeric polymerase, or anymodified polymerase that can effectuate the synthesis of a nucleic acidmolecule. Within this disclosure, a DNA polymerase may also include apolymerase, terminal transferase, reverse transcriptase, telomerase,and/or polynucleotide phosphorylase. Non-limiting examples ofpolymerases may include, for example, T7 DNA polymerase, eukaryoticmitochondrial DNA Polymerase y, prokaryotic DNA polymerase I, II, III,IV, and/or V; eukaryotic polymerase α, β, γ, δ, ε, η, ζ, ι, and/or κ; E.coli DNA polymerase I; E. coli DNA polymerase III alpha and/or epsilonsubunits; E. coli polymerase IV, E. coli polymerase V; T. aquaticus DNApolymerase I; B. stearothermophilus DNA polymerase I; Euryarchaeotapolymerases; terminal deoxynucleotidyl transferase (TdT); S. cerevisiaepolymerase 4; translesion synthesis polymerases; reverse transcriptase;and/or telomerase. Non-limiting examples of suitable thermostable DNApolymerases that may be used include Taq, Tfl, Tfi, Pfu, and Vent™ DNApolymerases, any genetically engineered DNA polymerases, any havingreduced or insignificant 3′-to-5′ exonuclease activity (e.g.,SuperScript™ DNA polymerase), and/or genetically engineered DNApolymerases (e.g., those having the active site mutation F667Y or theequivalent of F667Y (e.g., in Tth), AmpliTaq® FS, ThermoSequenase'),AmpliTaq® Gold, Therminator I, Therminator II, Therminator III,Therminator Gamma (all available from New England Biolabs, Beverly,Mass.), and/or any derivatives and fragments thereof. Other nucleic acidpolymerases may also be suitable as would be understood by one of skillin the art.

In another aspect, the present disclosure provides reaction mixtures forpolymerizing and/or amplifying a nucleic acid sequence of interest(e.g., a target sequence). In some embodiments, the reaction mixture mayfurther comprise a detectable label. The methods may also include one ormore steps for detecting the detectable label to quantitate theamplified nucleic acid. As used herein, the term “detectable label”refers to any of a variety of signaling molecules indicative ofamplification. For example, SYBR® Green and other DNA-binding dyes aredetectable labels. Such detectable labels may comprise or may be, forexample, nucleic acid intercalating agents or non-intercalating agents.As used herein, an intercalating agent is an agent or moiety capable ofnon-covalent insertion between stacked base pairs of a double-strandednucleic acid molecule. A non-intercalating agent is one that does notinsert into the double-stranded nucleic acid molecule. The nucleic acidbinding agent may produce a detectable signal directly or indirectly.The signal may be detectable directly using, for example, fluorescenceand/or absorbance, or indirectly using, for example, any moiety orligand that is detectably affected by proximity to double-strandednucleic acid is suitable such as a substituted label moiety or bindingligand attached to the nucleic acid binding agent. It is typicallynecessary for the nucleic acid binding agent to produce a detectablesignal when bound to a double-stranded nucleic acid that isdistinguishable from the signal produced when that same agent is insolution or bound to a single-stranded nucleic acid. For example,intercalating agents such as ethidium bromide fluoresce more intenselywhen intercalated into double-stranded DNA than when bound tosingle-stranded DNA, RNA, or in solution (see, e.g., U.S. Pat. Nos.5,994,056; 6,171,785; and/or 6,814,934). Similarly, actinomycin Dfluoresces in the red portion of the UV/VIS spectrum when bound tosingle-stranded nucleic acids, and fluoresces in the green portion ofthe UV/VIS spectrum when bound to double-stranded nucleic acids. And inanother example, the photoreactive psoralen4-aminomethyl-4-5′,8-trimethylpsoralen (AMT) has been reported toexhibit decreased absorption at long wavelengths and fluorescence uponintercalation into double-stranded DNA (Johnson et al. Photochem. &Photobiol., 33:785-791 (1981). For example, U.S. Pat. No. 4,257,774describes the direct binding of fluorescent intercalators to DNA (e.g.,ethidium salts, daunomycin, mepacrine and acridine orange,4′,6-diamidino-a-phenylindole). Non-intercalating agents (e.g., minorgroove binders as described herein such as Hoechst 33258, distamycin,netropsin) may also be suitable for use. For example, Hoechst 33258(Searle, et al. Nucl. Acids Res. 18(13):3753-3762 (1990)) exhibitsaltered fluorescence with an increasing amount of target. Minor groovebinders are described in more detail elsewhere herein.

Other DNA binding dyes are available to one of skill in the art and maybe used alone or in combination with other agents and/or components ofan assay system. Exemplary DNA binding dyes may include, for example,acridines (e.g., acridine orange, acriflavine), actinomycin D (Jain, etal. J. Mol. Biol. 68:21 (1972)), anthramycin, BOBO™-1, BOBO™-3,BO-PRO™-1, cbromomycin, DAPI (Kapuseinski, et al. Nucl. Acids Res.6(112): 3519 (1979)), daunomycin, distamycin (e.g., distamycin D), dyesdescribed in U.S. Pat. No. 7,387,887, ellipticine, ethidium salts (e.g.,ethidium bromide), fluorcoumanin, fluorescent intercalators as describedin U.S. Pat. No. 4,257,774, GelStar® (Cambrex Bio Science Rockland Inc.,Rockland, Me.), Hoechst 33258 (Searle and Embrey, Nucl. Acids Res.18:3753-3762 (1990)), Hoechst 33342, homidium, JO-PRO™-1, LIZ dyes,LO-PRO™-1, mepacrine, mithramycin, NED dyes, netropsin,4′,6-diamidino-α-phenylindole, proflavine, POPO™-1, POPO™-3, PO-PRO™-1,propidium iodide, ruthenium polypyridyls, S5, SYBR° Gold, SYBR° Green I(U.S. Pat. Nos. 5,436,134 and 5,658,751), SYBR® Green II, SYTOX® blue,SYTOX® green, SYTO® 43, SYTO® 44, SYTO® 45, SYTOX® Blue, TO-PRO®-1,SYTO® 11, SYTO® 13, SYTO® 15, SYTO® 16, SYTO® 20, SYTO® 23, thiazoleorange (Aldrich Chemical Co., Milwaukee, Wis.), TOTO™-3, YO-PRO®-1, andYOYO®-3 (Molecular Probes, Inc., Eugene, Oreg.), among others. SYBR®Green I (see, e.g., U.S. Pat. Nos. 5,436,134; 5,658,751; and/or6,569,927), for example, has been used to monitor a PCR reactions. OtherDNA binding dyes may also be suitable as would be understood by one ofskill in the art.

For use as described herein, one or more detectable labels and/orquenching agents may be attached to one or more primers and/or probes(e.g., detectable label). The detectable label may emit a signal whenfree or when bound to one of the target nucleic acids. The detectablelabel may also emit a signal when in proximity to another detectablelabel. Detectable labels may also be used with quencher molecules suchthat the signal is only detectable when not in sufficiently closeproximity to the quencher molecule. For instance, in some embodiments,the assay system may cause the detectable label to be liberated from thequenching molecule. Any of several detectable labels may be used tolabel the primers and probes used in the methods described herein. Asmentioned above, in some embodiments the detectable label may beattached to a probe, which may be incorporated into a primer, or mayotherwise bind to amplified target nucleic acid (e.g., a detectablenucleic acid binding agent such as an intercalating or non-intercalatingdye). When using more than one detectable label, each should differ intheir spectral properties such that the labels may be distinguished fromeach other, or such that together the detectable labels emit a signalthat is not emitted by either detectable label alone. Exemplarydetectable labels include, for instance, a fluorescent dye or fluorphore(e.g., a chemical group that can be excited by light to emitfluorescence or phosphorescence), “acceptor dyes” capable of quenching afluorescent signal from a fluorescent donor dye, and the like. Suitabledetectable labels may include, for example, fluorosceins (e.g.,5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Hydroxy Tryptamine (5-HAT); 6-JOE; 6-carboxyfluorescein (6-FAM); FITC;6-carboxy-1,4-dichloro-2′,7′-dichlorofluorescein (TET);6-carboxy-1,4-dichloro-2′,4′,5′,7′-tetrachlorofluorescein (HEX);6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE); Alexa fluor®fluorophores (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568,594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY® fluorophores(e.g., 492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568,564/570, 576/589, 581/591, 630/650-X, 650/665-X, 665/676, FL, FL ATP,FI-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-XSE), coumarins (e.g., 7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S,AMCA-X, ABQ, CPM methylcoumarin, coumarin phalloidin, hydroxycoumarin,CMFDA, methoxycoumarin), calcein, calcein AM, calcein blue, calcium dyes(e.g., calcium crimson, calcium green, calcium orange, calcofluorwhite), Cascade Blue, Cascade Yellow; Cy™ dyes (e.g., 3, 3.18, 3.5, 5,5.18, 5.5, 7), cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescentproteins (e.g., green fluorescent protein (e.g., GFP. EGFP), bluefluorescent protein (e.g., BFP, EBFP, EBFP2,Azurite, mKalama1), cyanfluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescentprotein (e.g., YFP, Citrine, Venus, YPet), FRET donor/acceptor pairs(e.g., fluorescein/tetramethylrhodamine, IAEDANS/fluorescein,EDANS/dabcyl, fluorescein/fluorescein, BODIPY® FL/BODIPY® FL,Fluorescein/QSY7 and QSY9), LysoTracker® and LysoSensor™ (e.g.,LysoTracker® Blue DND-22, LysoTracker® Blue-White DPX, LysoTracker®Yellow HCK-123, LysoTracker® Green DND-26, LysoTracker® Red DND-99,LysoSensor™ Blue DND-167, LysoSensor™ Green DND-189, LysoSensor™ GreenDND-153, LysoSensor™ Yellow/Blue DND-160, LysoSensor™ Yellow/Blue 10,000MW dextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines(e.g., 110, 123, B, B 200, BB, BG, B extra,5-carboxytetramethylrhodamine (5-TAN/IRA), 5 GLD, 6-Carboxyrhodamine 6G,Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red,Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine),Sulphorhodamine B can C, Sulphorhodamine G Extra, TAMRA(6-carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), WT),Texas Red, Texas Red-X, VIC and other labels described in, e.g., U.S.Patent Application Publication No. 2009/0197254 (incorporated herein byreference in its entirety), among others as would be known to those ofskill in the art. Other detectable labels may also be used (see, e.g.,U.S. Patent Application Publication No. 2009/0197254 (incorporatedherein by reference in its entirety)), as would be known to those ofskill in the art. Any of these systems and detectable labels, as well asmany others, may be used to detect amplified target nucleic acids.

Some detectable labels may be sequence-based (also referred to herein as“locus-specific detectable label”), for example 5′-nuclease probes. Suchprobes may comprise one or more detectable labels. Various detectablelabels are known in the art, for example (TaqMan® probes describedherein (See also U.S. Pat. No. 5,538,848 (incorporated herein byreference in its entirety)) various stem-loop molecular beacons (See,e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer,Nature Biotechnology 14:303-308 (1996)), stemless or linear beacons(See, e.g., PCT Publication No. WO 99/21881; U.S. Pat. No. 6,485,901),PNA Molecular Beacons™ (See, e.g., U.S. Pat. Nos. 6,355,421 and6,593,091), linear PNA beacons (See, e.g., Kubista et al., SPIE4264:53-58 (2001)), non-FRET probes (See, e.g., U.S. Pat. No.6,150,097), Sunrise®/Amplifluor® probes (U.S. Pat. No. 6,548,250),stem-loop and duplex Scorpions™ probes (Solinas et al., Nucleic AcidsResearch 29:E96 (2001) and U.S. Pat. No. 6,589,743), bulge loop probes(U.S. Pat. No. 6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250),cyclicons (U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (EpochBiosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleicacid (PNA) light-up probes (Svanvik, et al. Anal Biochem 281:26-35(2001)), self-assembled nanoparticle probes, ferrocene-modified probesdescribed, for example, in U.S. Pat. No. 6,485,901; Mhlanga et al.,Methods 25:463-471 (2001); Whitcombe et al., Nature Biotechnology.17:804-807 (1999); Isacsson et al., Molecular Cell Probes. 14:321-328(2000); Svanvik et al., Anal Biochem. 281:26-35 (2000); Wolffs et al.,Biotechniques 766:769-771 (2001); Tsourkas et al., Nucleic AcidsResearch. 30:4208-4215 (2002); Riccelli et al., Nucleic Acids Research30:4088-4093 (2002); Zhang et al., Acta Biochimica et Biophysica Sinica(Shanghai). 34:329-332 (2002); Maxwell et al., J. Am. Chem. Soc.124:9606-9612 (2002); Broude et al., Trends Biotechnol. 20:249-56(2002); Huang et al., Chem Res. Toxicol. 15:118-126 (2002); and Yu etal., J. Am. Chem. Soc. 14:11155-11161 (2001); QuantiProbes®(www.qiagen.com), HyBeacons® (French, et al. Mol. Cell. Probes15:363-374 (2001)), displacement probes (Li, et al. Nucl. Acids Res.30:e5 (2002)), HybProbes (Cardullo, et al. Proc. Natl. Acad. Sci. USA85:8790-8794 (1988)), MGB Alert (www.nanogen.com), Q-PNA (Fiandaca, etal. Genome Res. 11:609-611 (2001)), Plexor° (www.Promega.com), LUX™primers (Nazarenko, et al. Nucleic Acids Res. 30:e37 (2002)), DzyNAprimers (Todd, et al. Clin. Chem. 46:625-630 (2000)). Detectable labelsmay also comprise non-detectable quencher moieties that quench thefluorescence of the detectable label, including, for example, black holequenchers (Biosearch), Iowa Black® quenchers (IDT), QSY quencher(Molecular Probes), and Dabsyl and Dabcyl sulfonate/carboxylateQuenchers (Epoch). Detectable labels may also comprise two probes,wherein for example a fluorophore is on one probe, and a quencher is onthe other, wherein hybridization of the two probes together on a targetquenches the signal, or wherein hybridization on a target alters thesignal signature via a change in fluorescence. Exemplary systems mayalso include FRET, salicylate/DTPA ligand systems (see, e.g., Oser etal. Angew. Chem. Int. Engl. 29(10):1167 (1990)), displacementhybridization, homologous probes, and/or assays described in EuropeanPatent No. EP 070685 and/or U.S. Pat. No. 6,238,927. Detectable labelscan also comprise sulfonate derivatives of fluorescein dyes with SO₃instead of the carboxylate group, phosphoramidite forms of fluorescein,phosphoramidite forms of Cy5 (available for example from Amersham). Allreferences cited above are hereby incorporated herein by reference intheir entirety.

Other embodiments provide methods for inhibiting inactivation of apolymerase during a thermal cycling process by including therein a noveldetergent of Formula I. Also provided are methods for providing anenzyme having polymerase activity and at least one novel detergent ofFormula I and combining the same to form a mixture under conditions suchthat the polymerase activity of the enzyme is stabilized. The polymerasemay be any available to the skilled artisan, including but not limitedto those described herein. In certain embodiments, the polymerase isthermostable.

The detergents and methods described herein may be useful for detectingand/or quantifying a variety of target nucleic acids from a test sample.A target nucleic acid is any nucleic acid for which an assay system isdesigned to identify or detect as present (or not), and/or quantify in atest sample. Such nucleic acids may include, for example, those ofinfectious agents (e.g., virus, bacteria, parasite, and the like), adisease process such as cancer, diabetes, or the like, or to measure animmune response. Exemplary “test samples” include various types ofsamples, such as biological samples. Exemplary biological samplesinclude, for instance, a bodily fluid (e.g., blood, saliva, spinalfluid), a tissue sample, a food (e.g., meat) or beverage (e.g., milk)product, or the like. Expressed nucleic acids may include, for example,genes for which expression (or lack thereof) is associated with medicalconditions such as infectious disease (e.g., bacterial, viral, fungal,protozoal infections) or cancer. The methods described herein may alsobe used to detect contaminants (e.g., bacteria, virus, fungus, and/orprotozoan) in pharmaceutical, food, or beverage products. The methodsdescribed herein may be also be used to detect rare alleles in thepresence of wild type alleles (e.g., one mutant allele in the presenceof 10⁶-10⁹ wild type alleles). The methods are useful to, for example,detect minimal residual disease (e.g., rare remaining cancer cellsduring remission, especially mutations in the p53 gene or other tumorsuppressor genes previously identified within the tumors), and/ormeasure mutation load (e.g., the frequency of specific somatic mutationspresent in normal tissues, such as blood or urine).

Kits for performing the methods described herein are also provided. Asused herein, the term “kit” refers to a packaged set of relatedcomponents, typically one or more compounds or compositions. The kit maycomprise a pair of oligonucleotides for polymerizing and/or amplifyingat least one target nucleic acid from a sample, one or more noveldetergents (e.g., and/or conventional detergents, or a mixturecomprising any of the same), a biocatalyst (e.g., DNA polymerase) and/orcorresponding one or more probes labeled with a detectable label. Thekit may also include samples containing pre-defined target nucleic acidsto be used in control reactions. The kit may also optionally includestock solutions, buffers, enzymes, detectable labels or reagentsrequired for detection, tubes, membranes, and the like that may be usedto complete the amplification reaction. In some embodiments, multipleprimer sets are included. In one embodiment, the kit may include one ormore of, for example, a buffer (e.g., Tris), one or more salts (e.g.,KCl), glycerol, dNTPs (dA, dT, dG, dC, dU), recombinant BSA (bovineserum albumin), a dye (e.g., ROX passive reference dye), one or moredetergents (e.g., Dt4), one or more hot-start PCR mechanisms,polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), and/or gelatin(e.g., fish or bovine source). Other embodiments of particular systemsand kits are also contemplated which would be understood by one of skillin the art.

To more clearly and concisely describe and point out the subject matterof the present disclosure, the following definitions are provided forspecific terms, which are used in the following description and theappended claims. Throughout the specification, exemplification ofspecific terms should be considered as non-limiting examples.

The singular forms “a”, “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Approximating language, as usedherein throughout the specification and claims, may be applied to modifyany quantitative representation that could permissibly vary withoutresulting in a change in the basic function to which it is related.Accordingly, a value modified by a term such as “about” is not to belimited to the precise value specified. Where necessary, ranges havebeen supplied, and those ranges are inclusive of all sub-ranges therebetween.

In this disclosure, the use of the singular can include the pluralunless specifically stated otherwise or unless, as will be understood byone of skill in the art in light of the present disclosure, the singularis the only functional embodiment. Thus, for example, “a” may mean morethan one, and “one embodiment” may mean that the description applies tomultiple embodiments. The phrase “and/or” denotes a shorthand way ofindicating that the specific combination is contemplated in combinationand, separately, in the alternative.

It will be appreciated that there is an implied “about” prior to thetemperatures, concentrations, times, etc. discussed in the presentteachings, such that slight and insubstantial deviations are within thescope of the present teachings herein. Also, the use of “comprise”,“comprises”, “comprising”, “contain”, “contains”, “containing”,“include”, “includes”, and “including” are not intended to be limiting.It is to be understood that both the foregoing general description anddetailed description are exemplary and explanatory only and are notrestrictive of the invention.

Unless specifically noted in the above specification, embodiments in theabove specification that recite “comprising” various components are alsocontemplated as “consisting of” or “consisting essentially of” therecited components; embodiments in the specification that recite“consisting of” various components are also contemplated as “comprising”or “consisting essentially of” the recited components; and embodimentsin the specification that recite “consisting essentially of” variouscomponents are also contemplated as “consisting of” or “comprising” therecited components (this interchangeability does not apply to the use ofthese terms in the claims).

As used herein the terms “nucleotide” or “nucleotide base” refer to anucleoside phosphate. It includes, but is not limited to, a naturalnucleotide, a synthetic nucleotide, a modified nucleotide, or asurrogate replacement moiety or universal nucleotide (e.g., inosine).The nucleoside phosphate may be a nucleoside monophosphate, a nucleosidediphosphate or a nucleoside triphosphate. The sugar moiety in thenucleoside phosphate may be a pentose sugar, such as ribose, and thephosphate esterification site may correspond to the hydroxyl groupattached to the C-5 position of the pentose sugar of the nucleoside. Anucleotide may be, but is not limited to, a deoxyribonucleosidetriphosphate (dNTP) or a ribonucleoside triphosphate (NTP). Thenucleotides may be represented using alphabetical letters (letterdesignation). For example, A denotes adenosine (i.e., a nucleotidecontaining the nucleobase, adenine), C denotes cytosine, G denotesguanosine, T denotes thymidine, U denotes uracil, and I denotes inosine.N represents any nucleotide (e.g., N may be any of A, C, G, T/U, or I).Naturally occurring and synthetic analogs may also be used, includingfor example hypoxanthine, 2-aminoadenine, 2-thiouracil, 2-thiothymine,5-methylcytosine, N4-methylcytosine, 5,N4-ethencytosine,4-aminopyrrazolo[3,4-d]pyrimidine and6-amino-4-hydroxy[3,4-d]pyrimidine, among others. The nucleotide unitsof the oligonucleotides may also have a cross-linking function (e.g. analkylating agent).

As used herein, the term “oligonucleotide” or “polynucleotide” refers toan oligomer of nucleotide or derivatives thereof. The oligomers may beDNA, RNA, or analogues thereof (e.g., phosphorothioate analogue). Theoligomers may also include modified bases, and/or backbones (e.g.,modified phosphate linkage or modified sugar moiety). Non-limitingexamples of synthetic backbones that confer stability and/or otheradvantages to the oligomers may include phosphorothioate linkages,peptide nucleic acid, locked nucleic acid (Singh, et al. Chem. Commun.4:455-456 (1998)), xylose nucleic acid, and/or analogues thereof.Oligonucleotides may be any length “n.” For example, n may be any of 1,2, 4, 6, 8, 12, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 etc.number of nucleotides. The polynucleotide structure (N)_(n) representsan oligonucleotide consisting of n number of nucleotides N (e.g., (I)₈is representative of an oligonucleotide having the sequence IIIIIIII; or(A)₁₂ is representative of an oligonucleotide having the sequenceAAAAAAAAAAAA). Other types of oligonucleotides or polynucleotides mayalso be suitable for use as would be understood to one of skill in theart from this disclosure.

As used herein, the term “nucleic acid” refers to polymers ofnucleotides or derivatives thereof. As used herein, the term “targetnucleic acid” refers to a nucleic acid that is desired to be amplifiedin a nucleic acid amplification reaction. For example, the targetnucleic acid comprises a nucleic acid template.

As used herein, the term “sequence” refers to a nucleotide sequence ofan oligonucleotide or a nucleic acid. Throughout the specification,whenever an oligonucleotide/nucleic acid is represented by a sequence ofletters, the nucleotides are in 5′ to 3′ order from left to right. Forexample, an oligonucleotide represented by a sequence (I)_(n)(A)_(n)wherein n=1, 2, 3, 4 and so on, represents an oligonucleotide where the5′ terminal nucleotide(s) is inosine and the 3′ terminal nucleotide(s)is adenosine.

As used herein the term “reaction mixture” refers to the combination ofreagents or reagent solutions, which are used to carry out a chemicalanalysis or a biological assay. In some embodiments, the reactionmixture comprises all necessary components to carry out a nucleic acid(DNA) synthesis/amplification reaction. As described above, suchreaction mixtures may include at least one amplification primer pairsuitable for polymerizing and/or amplifying a nucleic acid sequence ofinterest and at least one detergent. As described above, a suitablereaction mixture may also include a “master mix” containing thecomponents (e.g., typically not including the primer pair) needed toperform an amplification reaction. The master mix may be combined withone or more detergents to form a reaction mixture. Other embodiments ofreaction mixtures are also contemplated herein as would be understood byone of skill in the art.

As used herein, the terms “reagent solution” or “solution suitable forperforming a DNA synthesis reaction” refer to any or all solutions,which are typically used to perform an amplification reaction or DNAsynthesis. They include, but are not limited to, solutions used in DNAamplification methods, solutions used in PCR amplification reactions, orthe like. The solution suitable for DNA synthesis reaction may comprisebuffer, salts, and/or nucleotides. It may further comprise primersand/or DNA templates to be amplified. One or more reagent solutions aretypically included in the reactions mixtures or master mixes describedherein.

As used herein, the term “primer” or “primer sequence” refers to a shortlinear oligonucleotide that hybridizes to a target nucleic acid sequence(e.g., a DNA template to be amplified) to prime a nucleic acid synthesisreaction. The primer may be a RNA oligonucleotide, a DNAoligonucleotide, or a chimeric sequence (e.g., comprising RNA and DNA).The primer may contain natural, synthetic, or modified nucleotides. Boththe upper and lower limits of the length of the primer are empiricallydetermined. The lower limit on primer length is the minimum length thatis required to form a stable duplex upon hybridization with the targetnucleic acid under nucleic acid amplification reaction conditions. Veryshort primers (usually less than 3 nucleotides long) do not formthermodynamically stable duplexes with target nucleic acid under suchhybridization conditions. The upper limit is often determined by thepossibility of having a duplex formation in a region other than thepre-determined nucleic acid sequence in the target nucleic acid.Generally, suitable primer lengths are in the range of about any of, forexample, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, (and so on) nucleotides long.

As used herein, “alkyl” refers to a hydrocarbon that is optionallylinear or branched, and may be fully saturated, mono- orpolyunsaturated. In addition, the term “alkyl,” as used herein, furtherincludes one or more substitutions at one or more carbon atoms of thehydrocarbon chain fragment.

As used herein, “aryl” refers to an aromatic moiety having a single ringor multiple condensed rings each of which is optionally andindependently substituted with H, halogen, cyano, azido, sulfonic acid,alkali or ammonium salt of sulfonic acid, carboxylic acid, biologicallycompatible salt of carboxylic acid, nitro, alkyl, perfluoroalkyl,alkoxy, alkylthio, amino, monoalkylamino, dialkylamino or alkylamido.

As used herein, “substituted” refers to a molecule wherein one or morehydrogen atoms are replaced with one or more non-hydrogen atoms,functional groups or moieties. For example, an unsubstituted nitrogen is—NH₂,while a substituted nitrogen is —NHCH₃. Exemplary substituentsinclude, but are not limited to, halo, e.g., fluorine and chlorine,alkyl, alkene, alkyne, sulfate, sulfone, sulfonate, amino, ammonium,amido, nitrile, alkoxy, phenoxy, aromatic, phenyl, polycyclic aromatic,and heterocycle.

Certain embodiments are further described in the following examples.These embodiments are provided as examples only and are not intended tolimit the scope of the claims in any way.

EXAMPLES Development of Novel Detergents

The novel detergents Dt1, Dt₂,Dt3, Dt4, Dt5, Dt6, Dt7, Dt8, Dt9, D10,Dt11, and Dt12 were developed using Process 1 described below:

Process 1

where R², R³, R⁴, R⁵, and n are as described hereinabove, and X isselected from the group consisting of H, CH₃, CH₂CH₃, CH₂(C₆H₅), andC(CH₃)₃. The novel detergents of Formula I may be, for example, ionic(e.g., cationic, anionic, zwitterionic).

As shown in Process 1, Compound A (1 eq.), methyltriphenoxyphosphoniumiodide (4 eq.), and N,N-dimethyl formamide (6 mL) were addedsequentially to a 50 mL aluminum foil covered round bottom flask. Thereaction was then allowed to stir for 3 days under argon atmosphere atroom temperature. After 3 days, the progress of the reaction wasmonitored using analytical LC-MS. The appearance of the intermediateproduct pattern will confirm its formation. This expected intermediateproduct (Intermediate B) was not isolated. To this intermediate productobtained from the previous step, added amino acid ester hydrochloridesalt (2 eq.) and Et₃N (2 eq.). The reaction mixture was heated for 3-4days at 65° C. The progress of the reaction was monitored usinganalytical LC-MS. The reaction mixture was cooled down to roomtemperature and was then concentrated down on the rotovapor toapproximately 2 mL. The concentrated crude mixture was the purified bypreparative HPLC. All the desired fractions were pooled and concentrateddown on the rotovapor to afford the desired product (the ionicdetergents Dt1, Dt3, Dt5, Dt7, Dt9, Dt11, Dt12). This product was thensubjected to hydrolysis reaction using 2N NaOH. The reaction mixture wasallowed to stir at room temperature until all the starting material wasconsumed as observed on analytical LC-MS, followed by neutralizationwith Amberlite® to afford the final zwitterionic products Dt₂,Dt4, Dt6,Dt10 and anionic product Dt8 as shown below:

where n is as described hereinabove. In certain embodiments, each n isindependently 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30.

Dt1 and Dt2 were tested for their ability to support nucleic acidamplification by a polymerase. Two different nucleic acid targets of 1kb and 3 kb (Rhod-1043 and Rhod-3637, respectively) were amplified byPCR using Taq polymerase in the presence of 0.1% NP-40 and 0.1% Tween®20 (control reactions), Dt1 or Dt2. As shown in FIGS. 1A and 1B, bothDt1 and Dt2 supported the amplification reactions in a comparable mannerto NP-40/Tween® 20. Dt1 supported amplification of the 1 kb amplicon(Rhod-1043) and the 3 Kb amplicon (Rhod-3637) when included in the 50 μlreaction at a concentration of between 0.008% and 0.0006%. Dt2 supportedamplification of the 1 Kb amplicon (Rhod-1043) and the 3 kb amplicon(Rhod-3637) when included in the 50 μl reaction at a concentration ofbetween 0.04% and 0.0001% (some amplification was observed at 0.0008%).

Dt1 was also tested using Rhodopsin gene primers to amplifyapproximately 4 Kb amplicons (Rhod-3920, Rhod-4181, and Rhod-4089) (FIG.2; “Storage B”: 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 50% glycerol, 1 mMDTT, distilled water). PCR conditions were 94° C. for two minutes; 35cycles of 15 seconds at 94° C., 30 seconds at 60° C., four minutes 30seconds at 72° C.; and extension for ten minutes at 72° C. Dt1 supportedamplification of Rhod-3920 and Rhod-4181 when included in the 50 μlreaction at a concentration of between 0.008% and 0.001%. Dt1 supportedamplification of Rhod-4089 when included in the 50 μl reaction at aconcentration of between 0.008% and 0.001%.

FIGS. 3A and 3B shows that 0.004% and 0.002% Dt1 is comparable to 0.004%NP-40/Tween® 20 in amplifying 0.1 to 1 Kb amplicons. FIGS. 4A and 4Bshows that 0.004% and 0.002% Dt1 is comparable to 0.004% NP-40/Tween® 20in amplifying 1-2 kb amplicons.

FIGS. 5A and 5B provides a comparison between Brij-58 and Dt1. As showntherein, Dt1 (0.04% to 0.006%) supports amplification in a comparablemanner to Brij-58 (0.04% to 0.0004%) or NP40/Tween® 20 (0.002%). Thedata indicates that this was not due to contamination of the Brij-58starting material used for modification.

FIG. 6 compares the the activity of Dt1 and Dt2. As shown therein, bothmodified detergents support amplification. Dt1 is shown to supportamplification when included in the reaction mixture at a concentrationof between 0.04% to 0.001%. Dt2 is shown to support amplification whenincluded in the reaction mixture at a concentration of between 0.04 to0.006%.

FIGS. 7A through 7D and 8A through 8D provide a comparison between Dt4and Tween® 20 in amplifying four different targets (B2M, GAPDH, RPLPO,and GUSB). As shown therein, amplification in the presence of 0.01% Dt4or 0.01% Tween® 20 provided similar results.

FIG. 9 provides a comparison between Dt4 and Tween 20 in amplifyingvarious different targets. As shown therein, amplification in thepresence of Dt4 or Tween® 20 provided similar results.

FIG. 10 illustrates the results of amplification in the presence of Dt1,Dt3, Dt5, Dt6 and Dt7. As shown therein, amplification was supported byeach detergent at the highest level by Dt4 followed by Dt1, although,under the reaction conditions of these experiment. Dt5 and Dt7 exhibitedsimilar activity, followed by Dt6.

FIGS. 11A and 11B, 12A and 12B, 13A and 13B, 14A and 14B, and 15A and15B show amplification plots comparing the activity of Dt4 to Brij-58and Tween® 20 at various concentrations in amplifying HPRT1 or PPIA. Asshown therein, Dt4 supports the amplification reaction in a comparablemanner to both Brij-58 and Tween® 20 in all concentrations tested(0.001% to 0.0001%).

FIGS. 16 through 18 illustrate that Dt4 may be used at a lowerconcentration than Tween® 20 in various reactions (e.g., 0.002% Dt4compared to 0.01% Tween® 20).

FIGS. 19 and 20 demonstrate that Dt4 is stable (e.g., it retains itsability to support amplification) in “5×” buffer (Tris (pH 8.0), KCl,and BSA) for at least two months.

FIGS. 21 and 22 provide a comparison of the ability of two Dt4 differentlots to support amplification of various targets, as compared to Tween20.

FIG. 23 illustrates that Dt4 supports amplification across a variety ofTaqMan assays.

All references cited within this disclosure are hereby incorporated byreference in their entirety. While certain embodiments have beendescribed in terms of the preferred embodiments, it is understood thatvariations and modifications will occur to those skilled in the art.Therefore, it is intended that the appended claims cover all suchequivalent variations that come within the scope of the followingclaims.

1. A compound of Formula I:

wherein: R¹ is a (C₈-C₁₆) alkyl or (C₈-C₁₆) substituted alkyl, R² and R³are each independently H or CH₃; R⁴ and R⁵ are each independently H,(CH₂)_(n)NH, (CH₂)_(n)N, or, alternatively, R³ is taken together with R⁵to form a 5- or 6-membered ring which is optionally substituted with atleast one (C₁-C₃₀) alkyl, (C₁-C₃₀) substituted alkyl, (C₁-C₃₀)heteroalkyl, (C₁-C₃₀) substituted heteroalkyl; and each n isindependently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or
 30. 2. (canceled) 3.The compound of claim 1, wherein the compound is selected from:

4.-27. (canceled)
 28. A nucleic acid amplification reaction mixturecomprising: a) at least one polymerase; b) dNTPs; and c) at least onecompound according to claim
 1. 29. The reaction mixture according toclaim 28, wherein said polymerase is thermostable.
 30. A method forpolymerizing a target nucleic acid comprising the steps of: a) combiningthe target nucleic acid with at least one polymerase and at least onecompound of claim 1 in a reaction mixture; and b) polymerizing thetarget nucleic acid. 31.-33. (canceled)
 34. The method of claim 30,wherein the reaction mixture further comprises at least one nucleic acidprimer and dNTPs.
 35. The method of claim 30, wherein polymerization ofthe target nucleic acid is detected.
 36. The method of claim 30, whereinpolymerization of the target nucleic acid is detected using a detectablelabel.
 37. The method of claim 36, wherein the detectable label is partof a primer or a probe.
 38. The method of claim 35, wherein thepolymerization of the target nucleic acid is quantitated.
 39. (canceled)40. The method of claim 30 wherein the thermostable polymerase isselected from the group consisting of Taq DNA polymerase, Tfi DNApolymerase, Tfl DNA polymerase, Pfu DNA polymerase, and Vent™ DNApolymerase, a polymerase having reduced 3′-to-5′ exonuclease activity,SuperScript™ DNA polymerase, a genetically engineered DNA polymerase, apolymerase having the active site mutation F667Y, a polymerase havingthe equivalent of active site F667Y, Tth polymerase, AmpliTaq®FS,ThermoSequenase™, Therminator I, Therminator II, Therminator III,Therminator Gamma, a derivative thereof, and a fragment thereof. 41.-57.(canceled)